Expression levels, biophysical properties and biological functions are three key features of an engineered protein. It is a challenge to preserve or improve expression level and biophysical properties of a protein while engineering its biological functions, as any introduced mutation may influence the structure of the protein, and this influence is by far still relatively unpredictable (Honegger et al, 2009).
Screening for protein candidates (PCs) with good expression levels and higher affinities has become more routine. Very high affinity binders are generated in many laboratories (Jonsson et al, 2008) and expression screening has made it possible to estimate the expression levels of a large number of proteins (Kery et al, 2003).
In contrast, engineering biophysical properties is more challenging. Strategies have been designed in all aspects of protein engineering to generate stable PCs. Single domain antibodies (sdAbs) derived from camelid heavy chain antibodies (Hamers-Casterman et al, 1993) are very stable molecules, but introduction of mutations (for humanization and affinity maturation) can lower their stabilities (Saerens et al, 2005). Careful design of libraries can greatly increase the proportion of PCs with good biophysical properties, but these libraries usually still contain significant percentage of proteins that are not satisfactory (Christ et al, 2007). One of the few exceptions is ankyrin repeats: most if not all reported protein binders built on small ankyrin domains seem to have good biophysical properties (Binz et al, 2004; Kohl et al, 2003). For evolving individual PCs, strategies such as molecular evolution based on sequence consensus (Lehmann et al, 2000) and introduction of potentially stabilizing residues (Ewert et al, 2003) have led to more stable proteins. In the selection process, the addition of high temperature (Jespers et al, 2004), extreme pH (Famm et al, 2008) and proteolytic (Ueda et al, 2004) pressures on PCs as well as selection on higher infectivity of phage displaying these PCs (Jespers et al, 2004; (Jespers et al, 2004 et al, 2005) have all led to successful selection of satisfactory binders. Despite these efforts, the challenge of routinely generating stable protein variants remains unmet. Another disadvantage of these approaches is their requirement for a specific molecular display platform, which is not suitable for many proteins.
It is noteworthy that the above approaches usually address only one of the three key features. In addition to the lack of research tools for generating proteins satisfying all aspects, PCs have to be purified in most cases for their characterization. This purification step renders characterization, even for less-challenging affinity screening, rather tedious work. Purifying and characterizing a large number of PCs thus becomes a significant limitation in protein engineering.
Screening methods for either expression levels (Kery et al, 2003), biophysical properties (Niesen et al, 2008; Woestenenk et al, 2003) or affinities (Leonard et al, 2007) are available, but few of the currently known approaches satisfies the requirement of both simplicity and high-throughput. Most such selection methods still require some level of protein purification, which is time-consuming. Additionally, the art-known methods do not allow screening of all key features outlined above.